Has anyone had success using the Dpn-I/Pfu method of introducing mutations? I'm trying to change 2 aa (3 bp change) that are 5 aa away from each other.
Any tips? I'm not using the kit... I plan to purchase the Pfu Ultra and DpnI seperately.
Hints/suggestions greatly appreciated!
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#2
btavshan
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Posted 05 May 2004 - 11:06 PM
It works brilliantly, even without the kit. The key is the design of the primers (use the guidelines in the stratagene manual, you can download it online) and their purity...it also helps to transform into a RecA- strain that's fairly competent (the Inoue method works fine). If you're paranoid about ensuring you're product is parental-DNA-free, you can use a 2 hour DpnI digestion. Other than that, it's a piece of cake.
The strategy we've used for designing primers for situations for yours is just using the web utility to design a single-mutation primer, and then adding the second mutation to that and elongating the primer on the appropriate side.
The strategy we've used for designing primers for situations for yours is just using the web utility to design a single-mutation primer, and then adding the second mutation to that and elongating the primer on the appropriate side.
Edited by btavshan, 05 May 2004 - 11:15 PM.
#3
unknownphd
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Posted 06 May 2004 - 07:19 AM
Thanks so much btavshan!
I had one more question... How big of a plasmid were you able to mutate? My plasmid is around 13kb and if I recall, stratgene's quickchange kit is for plasmids <8kb.
Also, did you ever encounter errors (i.e. pfu induced mutations?).
I had one more question... How big of a plasmid were you able to mutate? My plasmid is around 13kb and if I recall, stratgene's quickchange kit is for plasmids <8kb.
Also, did you ever encounter errors (i.e. pfu induced mutations?).
